• BMB Reports
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• 제43권6호
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• pp.419-426
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• 2010

Aeromonas hydrophila is a bacterial pathogen that infects a large number of eukaryotes, including humans. The UDP-galactose 4'-epimerase (GalE) catalyzes interconversion of UDP-galactose to UDP-glucose and plays a key role in lipopolysaccharide biosynthesis. This makes it an important virulence determinant, and therefore a potential drug target. Our earlier studies revealed that unlike other GalEs, GalE of A. hydrophila exists as a monomer. This uniqueness necessitated elucidation of its structure and active site. Chemical modification of the 6xHis-rGalE demonstrated the role of histidine residue in catalysis and that it did not constitute the substrate binding pocket. Loss of the 6xHis-rGalE activity and coenzyme fluorescence with thiol modifying reagents established the role of two distinct vicinal thiols in catalysis. Chemical modification studies revealed arginine to be essential for catalysis. Site-directed mutagenesis indicated Tyr149 and Lys153 to be involved in catalysis. Use of glycerol as a cosolvent enhanced the GalE thermostability significantly.

• 한국환경성돌연변이발암원학회지
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• 제22권3호
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• pp.175-182
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• 2002

Endocrine disruptors (EDs) are the chemicals that affect endocrine systems through activation or inhibition of steroid hormone response. It is necessary to have a good system to evaluate rapidly and accurately endocrine-disrupting activities of suspected chemicals and their degradation products. The key targets of EDs are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. We constructed a co-expression system of Gal4p DNA binding domain (DBD)- ligand binding domain of human estrogen receptor $\alpha$ or $\beta$, and Gal4p transactivation domain (TAD)-co-activator AIB-1, SRC-1 or TIF-2 in Saccharomyces cerevisiae with a chromosome-integrated lacZ reporter gene under the control of CYC1 promoter and Gal4p binding site (GAL4 upstream activating sequence, GAL4$_{UAS}$). Expression of this reporter gene was dependent on the presence of estrogen or EDs in the culture medium. We found that the two-hybrid system with combination of the hER$\beta$ LBD and co-activator SRC-1 was most effective in the xenoestrogen-dependent induction of reporter activity. The extent of transcriptional activation by those chemicals correlated with their estrogenic activities measured by other assay systems, indicating that this assay system is efficient and reliable for measuring estrogenic activity. The data in this research demonstrated that the yeast detection system using steroid hormone receptor and co-activator is a useful tool for identifying chemicals that interact with steroid receptors.s.

• Molecules and Cells
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• 제42권11호
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• pp.783-793
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• 2019

When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of $ATF6{\alpha}$ by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human $ATF6{\alpha}$ N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-$hATF6{\alpha}$ deletion variant was cleaved to liberate active transcription activator encompassing GV-$hATF6{\alpha}$ fragment which could translocate into the nucleus. The translocated GV-$hATF6{\alpha}$ fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-$hATF6{\alpha}$(333) represents an innovative tool to investigate regulated intramembrane proteolysis of $ATF6{\alpha}$. It can substitute active pATF6(N) binding motif-based reporter cell lines.

• Animal cells and systems
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• 제1권4호
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• pp.647-651
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• 1997

We have used a transient expression assay employing Drosophila S2 cells to study the transcriptional repression activity of a 27 amino acid residue-long repression domain FS1 which was generated by a frame-shift in a pair-rule gene, even-skipped of Drosophila melanogaster. In an attempt to define a minimal requirement for the repression activity, we constructed a series of truncation mutant forms of the FS1, fused to a heterologous GAL4 DNA-binding domain, and measured their activities. All of the mutant forms, including the GAL4-FS1 (5-23) which retains the smallest number (19) of amino acid residues of FS1, were found to repress an initiator, a minimal TATA-lacking promoter, in a GAL4-binding-site-dependent manner. These findings suggest that a 19 amino acid residue-long region, rich in proline and leucine residues, is a transcriptional repression domain and may interact with the general transcription machinery.

• KSBB Journal
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• 제10권3호
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• pp.343-348
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• 1995

LeI은 스테로이드를 통울에 투여하였을 때 분비가 촉진되어 항염증성 효과를 나타내는 calcium 의 존성 phospholipid 결합 단백질이다. S. cerevisiae는 대장균과 통물세포의 장점을 모두 가지고 있으므로 동물세포 유래의 이종 단백질의 분비 생산에 많이 이용되고 있다. 본 연구에서는 GAL10 promoter­p ppL-LCI유전자 LCI terminator로 구성된 pYGLPT5 로 LCI을 S. cerevisiae SEY2102에서 발현 분비시키고 각 분획으로 나누어 LCI양을 비교한 결과 protoplast 68.6 %. periplasmic 24 %, culture supernatant 7.4%로 분포하였다. pYGLPT5로 형질전환된 S. cereviswe 2102를 유가 배양한 결과, 최종적인 LCI의 생산량은 약 $500mg/\ell$ 였다. LCI은 N 말단 부근에 $CA^{++}$ 결합부위가 있으므로 이를 이용하여 hydroxylapatite column chromatography로 정 제하 였다. 배지로 분비된 34kDa LCI을 ultrafiltration 과 hydroxylapatite column chromatography 의 두 단계로 순도 99% 이상으로 정제할 수 있었다.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.316.1-316.1
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• 2002

Dermatan sulfate (DS) was isolated from eel skin (Anguilla japonica) bv actinase and endonuclease digeslions followed by ${\beta}$-elimination reaction and DEAE-Sephacel chromatography. DS was a major glycosaminoglycan in eel skin with 88% of the total uronic acid. The content of IdoA2S$\alpha$1longrightarrow4GalNAc4S sequence in eel skin. which is known to be a binding site to heparin cofactor II. was two times higher than that of dermatan sulfate from porcine skin. The anti-lla activity of eel skin dermatan sulfate mediated through heparin cofactor ll(NCL) was 25 units/mg. whereas DS from porcine skin shows 23.2 units/mg. The average molecular weight was determined as 14 kDa by gel chromatography on a TSKgel G3000SWXL column. Based on H1 NMR spectroscopy. we suggest that 3-sulfated and/or 2.3-sulfated ldoA residues are present in the chain.

• BMB Reports
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• 제35권3호
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• pp.313-319
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• 2002

N-Acetyl-$\beta$-D-hexosaminidase ($\beta$-HexNAc'ase) (EC 3.2.1.52) was purified from rice seeds (Oryza sative L. var. Dongjin) using ammonium sulfate (80%) precipitation, Sephadex G-150, CM-Sephadex, and DEAE-Sephadex chromatography, sequentially. The activities were separated into 7 fractions($F_1-F_7$) by CM-Sephadex chromatography. Among them, F6 was further purified to homogeneity with a 13.0% yield and 123.3 purification-fold. The molecular mass was estimated to be about 52 kDa on SDS-PAGE and 37.4 kDa on Sephacryl S-300 gel filtration. The enzyme catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-$\beta$-D-hexosaminide (pNP-GlcNAc) and p-nitrophenyl-N-acetyl-$\beta$-D-hexosaminide (pNP-GalNAc) as substrates, which are typical properties of $\beta$-HexNAc'ase. The ratio of the pNP-GlcNAc'ase activity to the pNP-GalNAc'ase activity was 4.0. However, it could not hydrolyze chitin, chitosan, pNP-$\beta$-glucopyranoside, or pNP-$\beta$-glucopyranoside. The enzyme showed $K_m$, $V_{max}$ and $K_{cat}$ for pNP-GlcNAc of 1.65 mM, $79.49\;mM\;min^{-1}$, and $4.79{\times}10^6\;min^{-1}$, respectively. The comparison of kinetic values for pNP-GlcNAc and pNP-GalNAc revealed that the two enzyme activities are associated with a single binding site. The purified enzyme exhibited optimum pH and temperature for pNP-GlcNAc of 5.0 and $50^{\circ}C$, respectively. The enzyme activity for pNP-GlcNAc was stable at pH 5.0-5.5 and $20-40^{\circ}C$. The enzyme activity was completely inhibited at a concentration of 0.1 mM $HgCl_$ and $AgNO_3$, suggesting that the intact thiol group is essential for activity. Chloramine T completely inhibited the activity, indicating the possible involvement of methionines in the mechanism of the enzyme.

• Journal of Microbiology and Biotechnology
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• 제30권9호
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• pp.1430-1435
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• 2020

Bacterial cellulose (BC) has outstanding physical and chemical properties, including high crystallinity, moisture retention, and tensile strength. Currently, the major producer of BC is Komagataeibacter xylinus. However, due to limited tools of expression, this host is difficult to engineer metabolically to improve BC productivity. In this study, a regulated expression system for K. xylinus with synthetic ribosome binding site (RBS) was developed and used to engineer a BC biosynthesis pathway. A synthetic RBS library was constructed using green fluorescent protein (GFP) as a reporter, and three synthetic RBSs (R4, R15, and R6) with different strengths were successfully isolated by fluorescence-activated cell sorting (FACS). Using synthetic RBS, we optimized the expression of three homologous genes responsible for BC production, pgm, galU, and ndp, and thereby greatly increased it under both static and shaking culture conditions. The final titer of BC under static and shaking conditions was 5.28 and 3.67 g/l, respectively. Our findings demonstrate that reinforced metabolic flux towards BC through quantitative gene expression represents a practical strategy for the improvement of BC productivity.